Bile Salts: The production of bile is an important function of human and animal liver hepatocytes and plays a crucial role in hepatobiliary and intestinal homeostasis and digestion. [de Buy Wenniger, Bile salts and cholestasis. Digest Liver Diseases 42(6), 409-18, 2010] Bile comprises a highly concentrated solution of bile salts—also known as conjugated bile acids—biliary lipids (phospholipids and cholesterol) and electrolytes. [Kis et al., Effect of membrane cholesterol on BSEP/Bsep activity: specificity studies for substrates and inhibitors, Drug Metabolism and Disposition 37, 1878-1886, 2009] Bile salts are synthesized in the liver via a series of metabolic steps starting from cholesterol. Bile salts and acids are secreted into bile and stored in the gallbladder for release. [Einarsson et al., Bile acid formation in primary human hepatocytes, World Journal of Gastroenterology 6(4), 522-525, 2000; Jansen and Faber, 2.3.6 Metabolism of bile acids in Hepatology—From Basic Science to Clinical Practice, Third edition, 2007, 174-181]
Bile salts or conjugated bile acids include glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, glycolithocholic acid and taurolithocholic acid (see FIG. 1).
After a meal, the gallbladder contracts, and stored bile is secreted into the intestinal tract where it plays a key role in the absorption of dietary lipids, fat-soluble vitamins, nutrients, and some drugs and drug candidates. In the intestine, approximately 90-95% of secreted bile salts are reabsorbed and returned to the liver and taken up there by hepatocytes—a process called enterohepatic circulation. [Jansen and Faber id page 174] Enterohepatic circulation serves an important physiological function not only for the recycling of bile salts and absorption of dietary lipids, fat-soluble vitamins, nutrients and some drugs and drug candidates, but also for the regulation of whole-body lipid metabolism. [Chiang, Bile acids: regulation of synthesis. Journal of Lipid Research 50, 1955-1966, 2009]
Bile salts are indispensable for the formation of bile flow; secretion of cholesterol and phospholipids from the liver, formation of mixed micelles that keep fat-soluble organic compounds in solution in the gut, promotion of the dissolution and hydrolysis of triglycerides by pancreatic enzymes, and act as signaling molecules in the regulation of enzymes and transporters of drugs and intermediary metabolism. [Jansen and Faber, id. page 178]
Biosynthesis of bile salts involves a multi-step process beginning with the initial oxidation of cholesterol by cytochrome P450 oxidase enzymes (also known as mixed function oxidases) present in human hepatocytes. (FIG. 2) [id. page 174, Chiang id. page 1955] Two main routes exist for the conversion of cholesterol to the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA):] a classic or neutral pathway involving initial oxidation by the cytochrome P450 CYP7A1 (cholesterol 7α-hydroxylase) and alternative or acidic pathway involving side chain hydroxylation with cytochrome P450 CYP27A1 (sterol 27-hydroxylase). [Jansen and Faber id. page 174] CYP7A1 is regarded as the rate-limiting enzyme in bile acid synthesis and deficiencies in animal models have been associated with severe liver failure. [Jansen and Faber id.] The CYP27A1 product is not a substrate for CYP7A1 but instead is oxidized by another cytochrome P450 enzyme named CYP7B1. From there, the neutral and acidic pathways overlap producing CDCA and CA. Other minor oxidative pathways may also contribute to bile acid synthesis. [Jansen and Faber id.] Most bile acids, including CA and CDCA, are conjugated to the amino acids glycine (G) and taurine (T) by two enzymes: bile acid:CoA synthase (BACS) and bile acid:amino acid transferase (BAT). [Chiang id. page 1957] These glycine and taurine bile acid conjugates act to decrease the toxicity and increase the aqueous solubility of unconjugated bile acids for secretion into bile. [Chiang id. page 1957] In the intestine, the glycine- and taurine-conjugated CA and CDCA can be deconjugated releasing CA and CDCA, which can be acted on by gut bacterial 7α-dehydroxylase to remove their 7α-hydroxy groups and thereby produce the secondary bile acids deoxycholic acid (DCA; 3α-12-dihydroxy CA) and lithocholic acid (LCA; 3α-monohydroxy). [Chiang id.] CA, CDCA, and DCA can be reabsorbed in the intestine and transported back to the liver to inhibit bile acid synthesis. Most of LCA is excreted in feces. The reabsorbed bile acids are further conjugated to amino acids producing the bile salts of CA, CDCA, and DCA.
Amino acid conjugated bile acids are termed conjugated bile acids or bile salts while non-amino acid conjugated bile acids are termed free. Bile acids and salts can be potentially toxic to cells and their concentrations under physiological conditions are tightly regulated. As mentioned above, bile acids are important and potent signaling molecules in the liver and intestine. Both free and conjugated bile acids bind to the ligand-binding domain of the nuclear transcription factor farnesoid X receptor (FXR; NR1H4), regulating FXR and associated gene transcription product FGF19 and ultimately regulating bile acid synthesis, excretion, and transport. [Chiang id. page 1956] Additionally, free and conjugated bile acids have been found to bind and activate pregnane X receptor (PXR; NR1I2) and vitamin D receptor (VDR; NR1I1). [Chiang id.]
The process of producing bile salts essentially results in the conversion of the hydrophobic cholesterol molecule into an amphipathic molecule that can serve physiologically as a detergent for absorption and transport of nutrients, fat-soluble vitamins, drugs, and other chemicals.
Bile salts have important acid-base properties, especially in the intestinal duodenum where pH values range from 3 to 5 units. [Costanzo, Physiology, 4th edition, Saunders/Elsevier, Philadelphia, Pa., 2010] Unconjugated bile acids have pKa values ranging near 7 pH units. In the duodenum, unconjugated bile acids are almost exclusively in the unionized protonated form and therefore are relatively insoluble in water and readily reabsorbed by the intestinal epithelium cells. Bile salts or conjugated bile acids have much lower pKa values ranging from 1 to 4 units whereas the conjugated bile salts are ionized or deprotonated in the duodenum, are more water soluble, and are more able to emulsify lipids and other non-water-soluble agents.
Bile salts or conjugated bile acids in the duodenum having been ionized, are not readily reabsorbed and can build up in concentrations to allow for formation of micelles and solubilized lipids which play significant roles in processes such as elimination of cholesterol from the body, removal of catabolites produced by the liver, and emulsifying lipids, fat-soluble vitamins, and some drugs and drug candidates. [Jansen and Faber id. page 178]
Hepatocytes: Hepatic parenchymal cells, or hepatocytes, are polyhedral or spherical in nature and account for approximately 60% of the cells in the liver; they represent 80% or more of the total liver volume. [de la Iglesia, Morphofunctional aspects of hepatic structure, Handbook of Drug Metabolism, edited by T. F. Woolf, 1999, page 83] Hepatocytes are polar in nature and one skilled in the art would recognize what is termed an apical (canalicular) membrane or domain and a basolateral (blood or sinusoidal domain) membrane or domain. The hepatocyte basolateral membrane or domain is involved in the uptake of drugs and xenobiotics into the cell, while the apical membrane or domain provides a route for intracellular produced bile salts to be excreted or transported into bile flow and eventually to the common bile duct for secretion into the intestine.
Hepatocytes have specialized transport systems or transcellular transporters located at the basolateral membrane and the apical membrane. [Morgan et al., Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development, Toxicological Sciences 118(2), 485-500, 2010] These hepatobiliary transporters maintain liver homeostasis by regulating intracellular exposure to endobiotic and xenobiotic chemicals. Transport systems comprising of specific transporter proteins have been extensively investigated. Transporters at the basolateral membrane are involved in hepatocellular uptake of various substrates from the blood and sinusoids, elimination to the blood and sinusoids, or both depending on the transporter. Transporters on the apical membrane, however, are exclusively efflux transporters, mediating secretion into the bile flow of various substrates including bile acids and salts. [Morgan et al., id. page 485]
Bile Salts Export Pump: BSEP (all capitalized letters reflect human transporter gene product) is a membrane-associated transporter protein located on the hepatocyte apical or canalicular membrane and is a member of the superfamily of ATP-binding cassette (ABC) transporter proteins, which are responsible for the extracellular transport or secretion of conjugated and unconjugated bile acids and salts into the bile canaliculi. [Kis et al., BSEP inhibition: in vitro screens to assess cholestatic potential of drugs, Toxicology In Vitro 26(8), 1294-9, 2012] BSEP is also known as ATP-binding cassette, sub-family B member 11, ABCB11, which is the protein product of the human ABCB11 gene (italics reflect the human gene). BSEP was first cloned in 1998 from rat and identified as the “sister of P-glycoprotein (sPGP)”, based on its close amino acid sequence similarity to P-glycoprotein. [Kis et al., Effect of membrane cholesterol on BSEP/Bsep activity: species specificity studies for substrates and inhibitors, Drug Metabolism and Disposition 37, 1878-1886, 2009, page 1878] BSEP displays higher transport affinity binding for tauro- and glycochenodeoxycholic acid and lower for taurocholic acid, glycocholic acid and tauroursodeoxycholic acid. [Jansen and Faber id. page 178] BSEP can transport to a limited extent unconjugated bile acids. [id.] BSEP, in addition to exporting bile salts, can also export some xenobiotics and drugs including pravastatin and vinblastine. [Morgan et al., id. page 485]
Rat and mouse orthologs of the human BSEP have similar amino acid sequences sharing 82% and 80%, respectively. [Yabuuchi H, et al, Cloning of the dog bile salt export pump (BSEP; ABCB11) and functional comparison with the human and rat proteins, Biopharmaceutical Drug Disposition, 29(8), 441-8, 2008] BSEP is specialized for transporting monovalent bile salts—taurine and glycine conjugates—through the canalicular membrane against a concentration gradient in an ATP-dependent manner. [Kis et al., id page 1878] BSEP transport of bile salts is a saturable process with Km values for bile salts in the low micromolar range. [Kis id.] The sensitivity to impairment in BSEP transport function appears to display species specificity. [Kis id.] Mutations in human BSEP can lead to progressive intrahepatic cholestasis and liver failure (see below).
Additional ABC transporters expressed at the apical and basolateral membrane include multidrug-resistance related protein MRP2 (ABCC2), breast cancer resistance protein BCRP (ABCG2, also known as MXR) and multidrug-resistance protein MDR1 (ABCB1, also known as P-glycoprotein). [Chandra and Brouwer, Pharmaceutical Research 21(5) 719-735, 2004]
In humans, the levels of the various transporter proteins are subject to genetic polymorphism in the encoding genes as well as in these transcription factors. Adverse drug reactions may be caused by genetic or disease-induced variations of transporter expression or drug-drug interactions at the level of these transporters. [Faber et al., Drug transport proteins in the liver, Advanced Drug Delivery Reviews 55(1), 107-24, 2003]
Drug-Induced Liver Injury (DILI): Drug-induced liver injury encompasses a spectrum of clinical diseases ranging from mild biochemical abnormalities to acute liver failure. [Hussanin and Farrington, Idiosyncratic drug-induced liver injury: an overview, Expert Opinion in Drug Safety 6(6), 673-84, 2007] Most frequently, the underlying mechanism of DILI is poorly understood. In some cases of DILI, the liver injury is categorized as idiosyncratic—unknown etiology. [Wolf et al., Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport, Toxicology In Vitro 24(1), 297-309, 2010; Lee, Drug-induced hepatotoxicity, New England Journal of Medicine 349(5), 474-85, 2003] The incidence of DILI induced hepatotoxicity in clinically marketed drugs is relatively rare, ranging from 1 in 5,000 to 1 in 10,000 or less. This is particularly true for DILI that results in severe liver injury leading to irreversible liver failure that can be fatal or require liver transplantation. DILI is a major cause of removal of approved drugs from the United States market resulting in removal of clinically significant therapeutics from patients in need of such therapy. [FDA Guidance for Industry: Drug-induced liver injury—premarketing clinical evaluation, July 2009; Ansede et al., An in vitro assay to assess transporter-based cholestatic hepatotoxicity using sandwich-cultured rat hepatocytes. Drug Metabolism and Disposition 38, 276-280, 2010] Additional consequences of DILI include class action lawsuits against the innovator company (with multi-million of dollar settlements), while adding additional time, expense, and uncertainty to the drug discovery and development process.
Because the modern drug development process requires extensive preclinical testing of drug candidates and subsequent clinical trials, drugs that do ultimately lead to DILI are rare. Drug candidates that display a toxic potential are usually removed from development and never reach the market. [FDA Guidance id.] Nevertheless, drugs that later result in DILI do get approved. Reasons for this may involve the relatively rare nature of the adverse event and that clinical trials are conducted in a closely controlled patient environment with a limited number of subjects for a limited time. Following marketing approval, the number of individuals administered a therapeutic agent will be much greater, periods of treatment may be much longer, and patients are less well monitored. Individuals display a wide variability in hepatic function and can differ greatly with respect to inherent hepatic metabolic function, environmental factors and co-medications. Risk factors for DILI include age, sex and genetic polymorphisms of drug-metabolizing enzymes such as cytochrome P450. In patients with human immunodeficiency virus, the presence of chronic viral hepatitis increases the risk of antiretroviral therapy hepatotoxicity. [Hussaini and Farrington, id. Abstract]
The relatively low incidence rate of DILI creates difficulties in detecting and diagnosing it, both for tests used and for numbers of patients needed. There is no clinical finding that indicates DILI with certainty, including liver biopsy. Because DILI may simulate any known liver disease, the histopathologic picture frequently is reported to be “compatible with” the clinical and laboratory information available, but is not often diagnostic. Therefore, the diagnosis of DILI is one of exclusion, in which sufficient clinical information must be gathered to rule out other possible causes of the abnormal findings. This diagnosis by exclusion requires collecting considerable data at the time of the acute clinical situation, a process that frequently is not well or thoroughly done, so that available information is inadequate to establish the likelihood of drug causality with any reasonable degree of confidence. [FDA Guidance, page 3-7]
In most controlled clinical trials, monitoring is done to detect hepatic injury by serum enzyme (typically aminotransferase) activity increases. Because risks associated with the new drug are unknown, caution has dictated that stopping rules be used to limit liver damage during the trial. For safety reasons, the drug may be stopped before the full implications of its possible toxicity can be determined. Extrapolation of such data, despite early withdrawal of the drug in many cases, is used to predict the likelihood of future severe toxicity when the drug is used clinically.
For interpreting data from patients exposed to drugs in clinical trials, there is a hierarchy of findings that indicate progressively severe liver injury, beginning with serum amino-transferase activities as the most frequently abnormal and most sensitive test. [FDA Guidance id] In many clinical trials of new drugs, up to 15% of study patients (or even more) may demonstrate mild elevations of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) activities. The threshold required to consider either more frequent monitoring of blood levels, or stopping the drug, is variously placed at twice the upper limit of the normal or reference range (2×ULN), 3×ULN, or 5×ULN. Monitoring is typically performed on a monthly basis but may be shortened to biweekly or weekly if elevations in serum enzyme levels are noted. According to the FDA guidance on drug-induced liver injury:
Discontinuation of treatment should be consideredALT or AST > 8 × ULNALT or AST > 5 × ULN for more than 2 weeksALT or AST > 3 × ULN and (TBL > 2 × ULN or INR > 1.5)ALT or AST > 3 × ULN with the appearance of fatigue, nausea, vomiting,right upper quadrant pain or tenderness, fever, rash, and/or eosinophilia(>5%)TBL—Total bilirubin LevelsINR—Increase plasma thrombin timeFDA Guidance for Industry - Drug induced liver injury: premarketing clinical evaluation, 2009, page 10
Levels of 10×ULN typically mandate immediate cessation and are considered more serious signals but still do not represent true tests of liver function. Yet great difficulties persist in making accurate attribution of causality as to whether the abnormalities seen are caused by DILI or by some other disorder. [FDA Guidance, pages 3-7, 10]
Even modest increases of serum total bilirubin concentration may represent the beginning of reduced bilirubin excretion capacity, provided Gilbert's syndrome and other unrelated causes of bilirubin elevation could be excluded. It is truly a function of the liver to clear plasma of bilirubin and excrete it into the bile. The late Hyman Zimmerman in 1978 and again in 1999 proposed that appearance of jaundice associated with drug-induced hepatocellular injury indicated possible mortality in 10 to 50% of patients showing that combination of abnormalities, based on his careful review of many clinical trials and literature reports. [FDA Guidance page 4]
Another commonly done test, the blood prothrombin time (or its derivative Internationalized Ratio, INR) may be useful as a liver function test (of protein synthesis). In acute liver failure caused by acetaminophen overdose, increases in INR may precede rises in total bilirubin levels. Thus, only a small decrement in liver function in pre-approval trials may provide a signal that additional and more severe cases may occur when larger numbers of patients are exposed. The full impact of this may not be realized until after approval for clinical use and marketing.
The condition of cholestasis occurs when bile and bile fluids cannot flow from the hepatocytes to the duodenum. The accumulation of bile salts in hepatocytes can lead to cellular apoptosis, necrosis and mitochondrial dysfunction. [Wolf et al., id. page 298] Cholestasis may result from physical obstructions—gallstones or tumors, or from metabolic disorders—drugs interfering with BSEP and other transporters.
BSEP inhibition and DILT: ATP-dependent transporters expressed on the apical plasma membrane domain of hepatocytes are important mediators of canalicular bile flow (see above). [Morgan et al., id. page 485] Impaired bile flow arising from genetically determined defects in transporters has been implicated in various inherited forms of cholestatic liver disease in humans. Genetic defects or mutations in BSEP are associated with at least three clinical forms of liver disease: (1) progressive familial intrahepatic cholestasis type 2 (PFIC2); (2) benign recurrent intrahepatic cholestasis type 2 (BRIC2); and (3) intrahepatic cholestasis of pregnancy. [Morgan et al., id. page 486] In the case of PFIC2, the condition has been associated with one or more polymorphisms in the genetic code for BSEP leading to inadequate BSEP function and associated liver injury. PFIC2 is characterized by progressive liver damage usually requiring transplantation while BRIC2 is characterized by intermittent and non-progressive cholestasis.
BSEP protein levels have been correlated with taurocholate transport activity in in vitro studies showing that patients with PFIC2 and BRIC2 gene mutations correlate with decreased protein expression. [Byrne et al., Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing, Hepatology 49, 553-567, 2009] Studies indicate that the extent of decrease in BSEP expression and function corresponds to disease outcome. [Morgan et al., id. page 486; Kagawa et al 2008]
Interference in BSEP function can lead to impaired hepatobiliary secretion of bile acids and salts leading to increased serum and tissue levels of bile acids and subsequent cellular mitochondrial damage, apoptosis (programmed cell death) and necrosis. [Maillette de Buy Wenniger, Bile salts and cholestasis, Dig Liver Dis. 42(6) 409-18, 2010]
Knockout mice have provided further insight into the complex interrelationships between expression of individual bile salt transporters, bile flow, and liver injury. Homozygous Bsep (mouse bile salt transporter protein) knockout mice were shown to develop severe cholestasis when fed a bile acid-enriched diet, whereas only mild cholestasis was observed when animals were fed a normal diet. This result has been attributed to adaptive changes in expression of other enzymes and transporters in Bsep (−/−) mice, which enable them to cope with the lack of functional Bsep expression unless challenged with a high dietary bile acid load. [Wang et al., Sever cholestasis induced by cholic acid feeding in knockout mice of sister of P-glycoprotein, Hepatology 38(6), 1489-99, 2003; Wang et al., Compensatory role of P-glycoproteins in knockout mice lacking the bile salt export pump, Hepatology 50(3), 948-56, 2009] Additional transporters have been implicated in cholestatic liver injury via studies undertaken in knockout mice include Mdr2 (the rodent ortholog of human MDR3). [Fickert et al., Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice, Gastroenterology 127(1), 261-74, 2004]
Therapeutic agents that interfere with BSEP function are often associated with liver liabilities in humans. [Morgan et al., id. page 486] Examples of drugs implicated in human liver injury where BSEP has been an implicated mechanism include bosentan (an endothelin antagonist for pulmonary arterial hypertension [PAH]), erythromycin estolate (a macrolide antibiotic), nefazodone (5-HT2 receptor antagonist for depression), CI-1034 (an experimental endothelin antagonist for pulmonary arterial hypertension [PAH]), and CP-724,714 (an experimental HER2 kinase inhibitor for oncology). [Morgan et al., id. page 486]
Agents that interfere with BSEP function and display liver injury in humans often are not associated with liver injury in preclinical animal investigations indicative of species differences. [Stieger et al., Role of the bile salt export pump, BSEP, in acquired forms of cholestasis, Drug Metabolism and Disposition 42, 437-445, 2009] This discrepancy in predicting liver toxicity of preclinical animal models is a significant concern for the pharmaceutical industry and increases the risk of unexpected liver injury in clinical development.
BSEP Inhibition and Drug-Drug Interactions: The potential for BSEP inhibitors to inhibit drug elimination was investigated in sandwich-cultured rat hepatocytes. [Jemnitz et al., Biliary efflux transporters involved in the clearance of rosuvastatin in sandwich cultured of rat hepatocytes, Toxicology In Vitro 24(2), 605-10, 2010] In this study, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor rosuvastatin, which is eliminated primarily unchanged by transporters, was used as a marker of transporter elimination. Various known inhibitors of elimination transporters were tested for effect on rosuvastatin elimination including the BSEP inhibitors cyclosporine A, glibenclamide and troglitazone. Results showed that cyclosporine A, glibenclamide and troglitazone interfered 32.6%, 29.3% and 20.6%, respectively, with rosuvastatin elimination. The investigators concluded a potential exists for drug-drug interactions with test agents that interfere with BSEP function.
BSEP Function Assays: In standard in vivo preclinical animal tests of drugs and drug candidates, agents found to interfere with BSEP function often don't induce significant liver injury; but nevertheless, have been associated with significant liver injury when administered to man. [Morgan et al., id. page 486] The inability of animal testing to predict the human hepatotoxicity potential of a drug, drug candidate, biological, food component, chemical, peptide, protein, oligonucleotide, DNA and RNA has led scientists skilled in the art to develop in vitro test model systems for BSEP function. These in vitro model systems include but not limited to:                (1) Sandwich-cultured hepatocyte (SCH) model prepared using primary animal or human hepatocytes;        (2) BSEP transfected Sf9 insect cell membrane vesicle models;        (3) Canalicular membrane vesicles (CMV) derived from rat and human whole liver; and        (4) Doubly-transfected with BSEP and sodium taurocholate co-transporting polypeptide (NTCP).        
Sandwich-Cultured Hepatocyte (SCH): Human and rat SCH is an in vitro model system that maintains many in vivo structural and functional characteristics of the hepatocyte, including canalicular and basolateral membrane domains, expression and localization of liver-specific proteins, and functional bile excretion into sealed canalicular networks. [Wolf et al., id. page 298] Human and rat SCH have been used to study the inhibitory effects of drugs including troglitazone, nefazodone, and antiretroviral agents.
Initial culturing methods employed for SCH led to realization of problems that diminish the ability of in vitro cultures to predict in vivo responses. [US2010035293A1 paragraph 0005] For example, culturing hepatocytes in the sandwich configuration form canalicular network(s) sealed by tight junctions, analogous to closed compartments, into which bile salts, bile acids and other components of bile are excreted. Due to the closed nature of the canalicular compartments, substances excreted from hepatocytes accumulate in these compartments. Over multiple days in culture, this results in cholestatic condition, wherein in bile is trapped in the bile ducts or compartments. Due to the “back-up” of the trapped bile salts and acids and other endogenous substances the hepatocytes may attempt to compensate by up-regulation or down-regulation of various transport proteins. In addition metabolic pathways also may be affected by the degree of cholestasis, leading to induction or inhibition of various enzymes including drug-metabolizing enzymes. [US2010035293A1 paragraph 0020] In an attempt to address this limitation, a process of pulsing the SCH at various time intervals by exposing the hepatocyte culture to a calcium-free buffer that releases the accumulated bile from the canalicular compartments. [US2010035293A1 paragraph 0021-0022] Such a method could reduce cholestasis in cultured hepatocytes and potentially be used as a model to predict the in vivo metabolism of compounds of interest. [US2010035293 paragraph 007] Further, such a method could allow for the development of models to evaluate the in vivo toxicity and biliary excretion of compounds of interest. [id.] Since SC human hepatocytes also retain metabolic capabilities, this model may allow for investigation of the interplay between many of the processes that take place in vivo. [Ghibellini et al., Methods to evaluate biliary excretion of drugs in human: an update review, Molecular Pharmacology 3(3), 198-211, 2006]
The pulsed SCH method assumes that regularly pulsing the in vitro hepatocyte culture will provide a system that more closely mimics in vivo hepatocytes, thereby providing a system that can not only assess transporter expression and function, but also be of use in evaluating hepatic toxicity and cholestasis. [US2010035293 paragraphs 44-47]
Nevertheless, the pulsed approach involves a complex series of steps involving using freshly isolated rat or human hepatocytes plated on gelled collagen coated 6-well plates and overlaying the cells with a layer of gelled collagen one day after plating to form the sandwich-culture configuration. [US2010035293 paragraph 51] The SCH culture is then pulsed at specific times for specific lengths with Hank's balanced salt solution (HBSS) with calcium (HBSS+Ca) or calcium-free HBSS (HBSS-Ca) followed by removal of the buffer. The frequency and length of pulsing can be important—incubation of HBSS-Ca for 30 minutes once per day or incubation of HBSS-Ca for 15 minutes twice per day. [US2010035293 paragraph 51] Clearly, the system is complex, has been developed with freshly prepared hepatocytes, and requires a high level of expertise to practice, and the requirement for frequent pulsing creates problems for contamination by microbes. The potential for differences in transport function between rat and human SCH in response to pulsing was not addressed. [Wolf et al., id. page 308]
Another issue with the SCH model is the limited number of sample wells available for experiments. In an attempt to address this problem, a modified method using an approach of cassette testing, incubations of multiple test agents in the same sample well, was described. [Wolf et al id.] The use of cassette testing of drug candidates, two to four drug candidates per incubation well, can lead to complex results—false positives and negatives, requiring follow-up testing of individual agents. The method has similar issues as described above along with a requirement for radiolabeled bile acids to measure transport activity. Because of the limited availability of fresh human hepatocytes and potential species differences with rat hepatocytes, the method is limited for any routine test agent-screening paradigm.
BSEP Transfected Sf9 Insect Cell Membrane Vesicles: The Sf9 system is widely used expression system for investigations of plasma membrane proteins properties. Because of its ability to express in significant amounts various membrane proteins, the system has been adapted to use with ABC transporters including human BSEP and various animal species Bsep. [Kis et al., id. page 1879; U.S. Pat. No. 8,129,197 column 2 lines 38-45]. Assay systems based on insect cell membrane preparations are generally stable, reliable, easy to handle and several assay formats are offered. [U.S. Pat. No. 8,129,197 id.] Nevertheless, insect cell membrane preparation assays differ when compared to mammalian cell based assays, which questions their value as useful and relevant assay systems for drug development. [U.S. Pat. No. 8,129,197 column 2 lines 46-50] Differences include high basal ATPase activity making transport assays less sensitive. [U.S. Pat. No. 8,129,197 column 2 lines 51-54] Differences between insect and mammalian membrane preparations have been observed in the activity of transporters and their sensitivity for drugs including sulfasalazine, topotecan, prazosin and methotrexate. [U.S. Pat. No. 8,129,197 column 2 lines 63-67] Some of these differences may be due to improper protein glycosylation and low Sf9 membrane cholesterol content. [Kis et al. id page 1883]
In an attempt to address this issue, Kis et al., added cholesterol to the BSEP/Bsep Sf9 transfected cell cultures to load the prepared membrane vesicles with additional cholesterol. [Kis et al., id page 1879] The optimized treatment increased the cholesterol 3- to 4-fold compared to untreated membrane vesicles. Inside-out vesicles are incubated at 37° C. for 5 min using 50 μg protein/well in the presence of 4 mM ATP and 2-μM total glycocholate (including 14C-glycocholate). The reaction is stopped by addition of ice-cold wash buffer with consecutive rapid filtration through Millipore Corporation (Billerica, Mass.) B-glass fiber filters of a 96-well filter plate. After washing five times with 200 μL of ice-cold wash buffer, the filters are dried, and the retained radioactivity measured in scintillation mixture. Species comparison of cholesterol loading showed the most pronounced effect on rat protein, whereas the activity of human BSEP was least affected by the treatment. [Kis et al., id. page 1881] In the assay, troglitazone and glibenclamide, compounds known to be cholestatic in humans, displayed species-specific inhibitory profiles. Results show that cholesterol loading makes BSEP/Bsep work more efficiently (higher Vmax), while not apparently changing the affinity (Km) for the transporter for most substrates tested. Nevertheless complexity of preparing the inside-out membrane vesicles and the differences between hepatocyte membranes and insects limits the utility of this method for predicting test agent inhibition potential. In addition, the method has issues of sensitivity, ease of use and requirement of radiolabeled bile salts.
A modified version of this method involving a taurochenodeoxycholate (TCDC) ATPase activity has been designed to be more user friendly, sensitive, and minus the radiolabeled bile salts. [Kis et al., Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs, Journal Biomolecular Screen 14(1), 10-15, 2009 (Abstract)] Comparison of TCDC transport measured by a vesicle transport assay and the TCDC-stimulated ATPase assay using cholesterol loaded transfected Sf9 insect inside-out vesicles showed ATPase assay to be sensitive for detection of transport function. A good rank order correlation was found between IC50 values measured in TCDC-stimulated mouse Bsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. The method may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay.
Saito et al 2009 describes many issues in preparing Sf9 inverted (inside-out) membrane vesicles including the timing of harvesting of Sf9 cells after baculovirus infection. [Saito et al., Technical pitfalls and improvements for high-speed screening and QSAR BSEP, The AAPS Journal, 11(3), 581-589, 2009, page 582] The study further highlights the importance of maintaining high integrity of the membrane vesicles used in transport assays.
In summary, the assay suffers from issues of transporter activity in membranes between insect and human/animal hepatocytes, complexity in preparing the transfected insect inside-out vesicles, the use of radiolabeled bile salts, and the inability to assess indirect affects (i.e. metabolism) of test agents on transport. Further, the assay does not allow for measuring formation of bile salts and test agent derived metabolites.
Canalicular Membrane Vesicles (CMV): The CMV has been used a model to study Bsep-mediated interactions isolated from humans and animals because this system contains all the relevant canalicular transporters at an expression level close to physiological. However, species specificity studies using CMVs from humans are difficult because of limited access to human samples and the complexity of dealing with polymorphisms. [Ghibellini et al., id. page 205; Kis et al., id. page 1883; Horikawa et al., Potential cholestatic activity of various therapeutic agents assessed by bile canalicular membrane vesicles isolated from rats and humans, Drug Metabolism and Pharmacokinetics 18, 16-22, 2003] Nevertheless, CMVs prepared from primary hepatocytes have distinct advantages over transfected systems: the native membrane environment is in place during isolation from the other cellular components, and all the hepatic transport systems are present. Membrane vesicles can be prepared, stored frozen, and used as needed; however, isolation of basolateral and canalicular fractions is labor intensive and complete purity is never achieved. [Ghibellini et al. id. page 205]
The CMV method was used to evaluate the potential of 15 therapeutic agents, known to cause cholestasis, to inhibit BSEP transports. The study was conducted using rat CMV. These results suggest that the majority of cholestasis-inducing drugs have a minimal inhibitory effect on rat BSEP and MRP2 although species differences in inhibitory potential should be considered, especially in the case of BSEP. [Horikawa et al. id.]
Doubly-Transfected Cell lines: A cell-based assay system in which a liver uptake transporter (human sodium taurocholate co-transporting polypeptide [NTCP]) is constitutively expressed together with a liver export pump (ABC transporter—human BSEP) in the polarized canine kidney cell line MDCKII. [US2007/0287167 paragraph 0007] The resulting dog kidney cell line, therefore, contains human NTCP together with human BSEP. The cells are cultivated in 6-well filter inserts on a porous filter membrane that separates the basolateral membrane domain from the apical membrane domain. Test compounds are added to either the apical domain compartment or basolateral domain compartment for transcellular (vectorial) transport measurements in both directions. Samples are taken from each compartment at designated time periods. Substrates of the combination expressed transporters display significant net transport from basolateral to the apical compartment when compared to un-transfected cell. This method can be used to assess the test compound potential to participate in hepatobiliary elimination. The method can also be used to investigate drug-drug interaction potential between two test compounds. [id., paragraph 0010] Nevertheless, the system differs significantly from that of human hepatocytes.